Global absolute quantification of a proteome: Challenges in the deployment of a QconCAT strategy

In this paper, we discuss the challenge of large-scale quantification of a proteome, referring to our programme that aims to define the absolute quantity, in copies per cell, of at least 4000 proteins in the yeast Saccharomyces cerevisiae. We have based our strategy on the well-established method of stable isotope dilution, generating isotopically labelled peptides using QconCAT technology, in which artificial genes, encoding concatenations of tryptic fragments as surrogate quantification standards, are designed, synthesised de novo and expressed in bacteria using stable isotopically enriched media. A known quantity of QconCAT is then co-digested with analyte proteins and the heavy:light isotopologues are analysed by mass spectrometry to yield absolute quantification. This workflow brings issues of optimal selection of quantotypic peptides, their assembly into QconCATs, expression, purification and deployment.     

Assessment of genetic diversity and structure in cocoa trees (<Emphasis Type="Italic">Theobroma cacao</Emphasis> L.) in Côte d’Ivoire with reference to their susceptibility to Cocoa swollen shoot virus disease (CSSVD)

Resistance to Cocoa Swollen Shoot Virus disease (CSSVD) is becoming an increasingly important criterion for selection of new cocoa cultivars in Côte-d’Ivoire, where the disease resurfaced since 2003. This virus can seriously affect the yield of trees with a loss of 25%, 1 year after infection, to around 100% 3 years after. In order to find tolerant plant material, 337 farm accessions have been collected on fields affected by CSSVD, according to the status of accessions potentially tolerant (APT) or susceptible (APS). Both phenotypic groups were genotyped using 30 microsatellite markers (SSR) in the presence of representative clones of the ten genetic groups of cocoa. This study revealed 214 alleles with the set of primer pairs used. The number of alleles per locus was between 3 and 16 with an average of 7.13 alleles per locus. The results showed a high contribution of genetic diversity within population (H_s?=?0.51) to the total genetic diversity (H_t?=?0.53) for the two studied groups. There was no significant difference between tolerant and susceptible groups (Fst?=?0.05). These results suggest that APT could be a potential genetic reservoir for other traits of interest associated with virus resistance. The phylogenetic tree, as the STRUCTURE analysis of Ivorian cocoa population, showed a distribution of individuals following four groups marked by a high contribution of group 4 (Nanay, Maranon, Guiana) followed by group 2 (Criollo), and group 1 (Amelonado), and a lower contribution of group 3 (Iquitos, Purus, Nacional, Curaray, Contamana).     

Divergent in shape and convergent in function: Adaptive evolution of the mandible in Sub‐Antarctic mice

Convergent evolution in similar environments constitutes strong evidence of adaptive evolution. Transported with people around the world, house mice colonized even remote areas, such as Sub‐Antarctic islands. There, they returned to a feral way of life, shifting towards a diet enriched in terrestrial macroinvertebrates. Here, we test the hypothesis that this triggered convergent evolution of the mandible, a morphological character involved in food consumption. Mandible shape from four Sub‐Antarctic islands was compared to phylogeny, tracing the history of colonization, and climatic conditions. Mandible shape was primarily influenced by phylogenetic history, thus discarding the hypothesis of convergent evolution. The biomechanical properties of the jaw were then investigated. Incisor in‐lever and temporalis out‐lever suggested an increase in the velocity of incisor biting, in agreement with observations on various carnivorous and insectivorous rodents. The mechanical advantage related to incisor biting also revealed an increased functional performance in Sub‐Antarctic populations, and appears to be an adaptation to catch prey more efficiently. The amount of change involved was larger than expected for a plastic response, suggesting microevolutionary processes were evolved. This study thus denotes some degree of adaptive convergent evolution related to changes in habitat‐related changes in dietary items in Sub‐Antarctic mice, but only regarding simple, functionally relevant aspects of mandible morphology.     

Proline accumulation in canola leaf discs subjected to osmotic stress is related to the loss of chlorophylls and to the decrease of mitochondrial activity

Many stress studies use simplified experimental models like leaf discs, but the validity of such approaches remains a matter of debate, especially when attempts are made to obtain suitable biotests. We report here the analysis of the resistance of canola leaf discs to osmotic stress, as related to the accumulation of proline. For that purpose, the explanted tissues were incubated for 24 h under continuous light and at 28°C on polyethylene glycol solutions of external potentials (Π_ext) ranging from −0.1 to −8.0 MPa. Proline content, water deficit and chlorophyll content were quantified. The cell membrane stability, which is a structural component of viability, was assessed according to electrolyte leakage. Biomembrane oxidative damage was measured through the production of malondialdehyde and the mitochondrial activity was quantified by assessing the ability of the tissues to reduce 2,3,5-triphenyltetrazolium chloride. Although the water deficit of the tissues reached high values (i.e. up to 70%), the cell membrane stability remained high. Furthermore, the oxidative damage to biomembranes was quite low, even in highly dehydrated tissues. In contrast, osmotic stress induced a significant decrease in the chlorophyll content and a strong accumulation of proline. These two processes each reached a maximum at a Π_ext of −3.0 MPa, although the tissues appeared to be viable at even lower Π_ext, suggesting they are not responsible for primary resistance. The mitochondrial activity was strongly decreased in the stressed leaf discs, although this activity was at least partially restored in tissues incubated for 24 h on a non-stressing medium. It appears that the cell components of the osmotic stress resistance assessed observed in this study do not reflect the modes of resistance of an intact plant.     

End buds: non-ampullary electroreceptors in adult lampreys

Summary Shared anatomical and physiological characters indicate that the low-frequency sensitive electrosensory system of lampreys is homologous with those of non-teleost fishes and amphibians. However, the ampullary electroreceptor organs which characterize all of these gnathostomes are not found in lampreys. Experimental anatomical and physiological studies reported here demonstrate that the epidermal end buds are the electroreceptors of adult lampreys.End buds, consisting of both sensory and supporting cells, are goblet-shaped with the top (25–60 m diameter) at the epidermal surface and the stem directed toward the dermis (Fig. 1A). Short lines or clusters of 2–8 end buds (Fig. 1B) are distributed over both trunk and head. Injections of horseradish peroxidase (HRP) into vitally-stained end buds labeled anterior lateral line afferents terminating in the ipsilateral dorsal nucleus (Fig. 2A) — the primary electrosensory nucleus of the lamprey medulla. Conversely, after HRP injection into the dorsal nucleus HRP-filled fibers and terminals were present on ipsilateral end buds (Fig. 2B).End buds are usually not visible without staining. However, in adult sea lampreys the presence of end buds was histologically confirmed in skin patches containing the receptive fields of electroreceptor fibers recorded in the anterior lateral line nerve. Additionally, in the rare instance of two silver lampreys in which end buds were visible without staining, electrosensory activity indistinguishable from that of the primary electroreceptor afferents was recorded from the end bud surface (Figs. 3, 4).End buds were initially characterized as chemoreceptors (Johnston 1902) but were later correctly advanced as lateralis receptors based on the presence of presynaptic dense bodies in the receptor cells (Whitear and Lane 1981). Unlike all other low-frequency electroreceptors, end buds lack canals. The receptor cells contact the epidermal surface and possess apical microvilli rather than the kinocilium of most gnathostomes with homologous electrosensory systems of the primitive (non-teleost) type.Larval lampreys and newly transformed adults lack end buds although at least the latter are electroreceptive. End buds, therefore, may be the form taken by electroreceptors only in the final portion of a lamprey's life.     

Amino acid sequence of human plasma apolipoprotein C-II from normal and hyperlipoproteinemic subjects

The complete amino acid sequence of human plasma apolipoprotein C-II isolated from normal individuals and a subject with type V hyperlipoproteinemia has been determined. Apo-C-II contains 79 amino acids with the following amino acid composition: Asp4, Asn1, Thr9, Ser9, Glu7, Gln7, Pro4, Gly2, Ala6, Val4, Met2, Ile1, Leu8, Tyr5, Phe2, Lys6, Arg1, Trp1. Cleavage of apo-C-II by cyanogen bromide produced three peptides designated as CB-1 (9 residues), CB-2 (51 residues), and CB-3 (19 residues). Two peptides, CT-1 (50 residues) and CT-2 (29 residues), which overlapped the cyanogen bromide peptides, were obtained by tryptic digestion of citraconylated apo-C-II at the single arginine residue. The amino acid sequence of apo-C-II was obtained by the automated phenyl isothiocyanate degradation of intact apo-C-II and the peptides produced by cleavage of apo-C-II by cyanogen bromide and trypsin. Phenylthiohydantoins were identified by high performance liquid chromatography and chemical ionization-mass spectrometry. The amino acid sequence of apo-C-II from the normal subject was identical with the apo-C-II isolated from the hyperlipoproteinemic subject.